Tumor activated prodrugs

ABSTRACT

The instant invention provides compositions comprising a prodrug, the prodrug comprising a therapeutically active drug; and a peptide selected from the group consisting of the sequences: Ser-Ser-Lys-Tyr-Gln (SEQ ID NO:1);Gly-Lys-Ser-Gln-Tyr-Gln (SEQ ID NO:2); and Gly-Ser-Ala-Lys-Tyr-Gln (SEQ ID NO:3) wherein the peptide is linked to the therapeutically active drug to inhibit the therapeutic activity of the drug, and wherein the therapeutically active drug is cleaved from the peptide upon proteolysis by an enzyme having a proteolytic activity of prostate specific antigen (PSA). The invention further provides methods of making and using the claimed compositions.

GOVERNMENT FUNDING

This invention was made, in whole or in part, by grant P50CA58236 fromthe National Cancer Institute. Accordingly, the Government may havecertain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the targeted activation ofbiologically active materials to cells that produce prostate specificantigen (PSA) and more specifically to PSA-cleavable peptides thatactivate therapeutic drugs.

BACKGROUND OF THE INVENTION

There is currently no effective therapy for men with metastatic prostatecancer who relapse after androgen ablation, even though numerous agentshave been tested over the past thirty years. Prolonged administration ofeffective concentrations of standard chemotherapeutic agents is usuallynot possible because of dose-limiting systemic toxicities.

Prostate specific antigen (PSA) is a 33,000 kDa single chainglycoprotein first characterized from human prostate tissue. PSA issynthesized and secreted as a unique differentiation product of theprostatic glandular cells, both from normal and cancerous cells. Lowlevels of PSA are detected in normal and cancerous breast tissue also.

Prostate Specific Antigen (PSA) is a chymotrypsin-like serine proteasethat is measurable in the blood and is used as a clinical test to detectprostate cancer and follow response to therapy. However, PSA is notactive in the blood and is only active within tumor sites and in thenormal prostate tissue. The concept of capitalizing upon the prostatespecific expression of the protease PSA to target therapeutic agents toprostate cancer sites was first proposed in 1992. Since that time,considerable development, research and systematic effort have beenapplied to bring that idea to fruition. These efforts have resulted inidentification of an initial PSA-activated pro-drugs which have beendescribed in detail elsewhere (see, for example, U.S. Pat. No.6,410,514).

Thapsigargin (TG) is an sesquiterpene-γ-lactone available by extractionfrom the seeds and roots of the umbelliferous plant Thapsia garganica L.Thapsigargin selectively inhibits the sarcoplasmic reticulum (SR) andendoplasmic reticulum (ER) Ca²⁺ -ATPase (SERCA) pump, found in skeletal,cardiac, muscle and brain microsomes. The apparent dissociation constantis 2.2 pM or less.

TG operates by what is believed to be a unique method of killing cells.TG induced inhibition of the SERCA pump leads to depletion of the ERCa2+ pool. This depletion apparently results in the generation of asignal, possibly from an ER-derived diffusible messenger, so that theplasma membrane is more permeable to extracellular divalent cations. Theresulting influx of these cations is responsible for the death of cells.

TG is poorly soluble in water, does not possess cell specificity, and isable to kill quiescent G_(o) cells. For these reasons, unmodified TGwould be difficult to administer and deliver systemically withoutsignificant non-specific host toxicity.

Accordingly, the need exists for improved tumor-activated pro-drugs forthe treatment of cell proliferative disorders, e.g., cancer.

SUMMARY OF THE INVENTION

The present invention provides peptides consisting of or comprisingSSKYQ that include a cleavage site for prostate specific antigen (PSA)and other enzymes with the same activity and proteolytic specificity asPSA. The invention also provides analogs, derivatives and conservativevariations of these peptides.

The invention also provides a therapeutic prodrug composition,comprising a therapeutic drug linked to a peptide of the invention whichis specifically cleaved by PSA. The linkage substantially inhibits thenon-specific toxicity of the drug, and cleavage of the peptide releasesthe drug, activating it or restoring its non-specific toxicity. Theinvention provides therapeutic prodrug compositions comprising a peptideof the invention, e.g., SSKYQ, and a thapsigargin or a thapsigarginderivative. The thapsigargins are a group of natural products isolatedfrom species of the umbelliferous genus Thapsia. The term“thapsigargins” has been defined by Christensen, et al., Prog. Chem.Nat. Prod., 71 (1997) 130-165. These derivatives contain a means oflinking the therapeutic drug to carrier moieties, including peptides andantibodies. The peptides and antibodies can include those whichspecifically interact with antigens including PSA. The interactions caninvolve cleavage of the peptide to release the therapeutic analogs ofsesquiterpene-γ-lactones.

Prodrug composition comprising a PSA cleavable peptide and a therapeuticdrug have been previously disclosed (see, for example, U.S. Pat. No.6,410,514). The prodrug compositions disclosed herein have improvedcharacteristics as compared to the compositions previously described.Unexpectedly, the prodrug compositions described herein have increased,hydrolysis by PSA, increased antitumor efficacy, and increasedgeneration of therapeutic drug at the site of the tumor as compared tothe compositions previously described.

The invention also provides a method for treating cell proliferativedisorders, including those which involve the production of PSA, insubjects having, or at risk of having such disorders. The methodinvolves administering to the subject a therapeutically effective amountof the composition of the invention.

The invention also provides a method of producing the prodrugcomposition of the invention. In another embodiment, the inventionprovides a method of detecting PSA activity in tissue. In yet anotherembodiment, the invention provides a method of selecting appropriateprodrugs for use in treating cell proliferative disorders involvingPSA-production.

The invention also provides a method for detecting a cell proliferativedisorder associated with PSA production in a tissue of a subject,comprising contacting a target cellular component suspected of having aPSA associated disorder, with a reagent which detects enzymaticallyactive PSA.

The invention also provides a method of determining PSA activity in aPSA-containing sample, comprising contacting the sample with adetectably labeled peptide of the invention which is specificallycleaved by PSA for a period of time sufficient to allow PSA to cleavethe peptide, detecting the detectable label to yield a detection level,which is then compared to the detection level obtained by contacting thesame detectably labeled peptide with a standard PSA sample of knownactivity.

The invention also provides a method of imaging soft tissue and/or bonemetastases which produce PSA, comprising administering a lipophilicimaging label linked to a peptide of the invention which is specificallycleaved by PSA to a subject having or suspected of having aPSA-associated cell proliferative disorder, allowing PSA to cleave thepeptide, allowing the lipophilic imaging label to accumulate in thetissue and/or bone, allowing the subject to clear the uncleaved peptide,and imaging the subject for diagnostic purposes.

Unless otherwise defined, all technical and scientific terms used hereinhave the ordinary meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other reference materials mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structures of (A) Thapsigargin and L12ADT;(B) Mu-HSSKLQ//L12ADT; and (C) Mu-SSKYQ//L12ADT (the // represents thelocation of PSA cleavage).

FIG. 2 depicts the hydrolysis of prodrugs (100 μM final concentration)by PSA (10 μg/ml) in PSA buffer (50 mM Tris, 0.1 M NaCl, pH 7.8).Release of product L12ADT was determined by liquid chromatography/massspectrometric (LC/MS) analysis and calculation of area under the curve(AUC).

FIG. 3 depicts the antitumor effect of prodrugs compared to vehiclecontrol against CWR22H xenografts following 10 daily injections of 0.1μmole/dose.

FIG. 4 depicts the trough levels of prodrugs and L12ADT following 10consecutive intravenous treatments at the indicated dose levels.

DETAILED DESCRIPTION

The invention provides novel peptides consisting of or comprising theamino acid sequence Ser-Ser-Lys-Tyr-Gln (SSKYQ) which contain a cleavagesite specific for prostate specific antigen (PSA). In other preferredembodiments of the invention are provide novel peptides consisting of orcomprising the amino acid sequence Gly-Lys-Ser-Gln-Tyr-Gln (GKSQYQ) andGly-Ser-Ala-Lys-Tyr-Gln (GSAKYQ). These peptides are efficiently andspecifically cleaved by PSA. The peptide is useful for substantiallyinhibiting the non-specific toxicity of the therapeutic agents prior tothe agents contacting a tissue containing PSA. The invention furtherprovides prodrugs comprising sesquiterpene-γ-lactone analogs linked to apeptide of the invention as described herein. The linkage substantiallyconverts the derivative into an inactive prodrug. The compositions donot show significant non-specific toxicity, but in environments wherePSA is found, the composition becomes activated when peptide is cleaved,releasing the therapeutic drug, which regains its non-specific toxicity.

PSA-Specific Peptides

As used herein, the term “prostate specific antigen” (PSA) meansprostate specific antigen, as well as all other proteases that have thesame or substantially the same proteolytic cleavage specificity asprostate specific antigen. As used herein, “sufficiently toxic” refersto therapeutic drugs which display nonspecific toxicity toward cellswith an LC₅₀ concentration that is at least 3 times lower than the LC₅₀concentration of the prodrugs of the invention, more preferably at least20 times lower, and therapeutic drugs most preferably have an LC₅₀concentration that is at least 100 times lower than the LC₅₀concentration of the prodrugs of the invention. The term “contacting”refers to exposing tissue to the peptides, therapeutic drugs or prodrugsof the invention so that they can effectively inhibit cellularprocesses, or kill cells. Contacting may be in vitro, for example byadding the peptide, drug, or prodrug to a tissue culture to test forsusceptibility of the tissue to the peptide, drug or prodrug. Contactingmay be in vivo, for example administering the peptide, drug or prodrugto a subject with a cell proliferative disorder, such as prostate orbreast cancer or benign prostatic hypertrophy. By “polypeptide” is meantany chain of amino acids, regardless of length or post-translationalmodification (e.g., glycosylation or phosphorylation). As writtenherein, amino acid sequences are presented according to the standardconvention, namely that the amino terminus of the peptide is on theleft, and the carboxy terminus on the right. In one aspect, theinvention features a peptide, e.g., SSKYQ, that includes a cleavagerecognition site for PSA or an enzyme having a proteolytic activity ofPSA. The preferred amino acid sequences of the invention is linear.

The cleavage site recognized by PSA is flanked by the amino acidsequences SSKYQ, GKSQYQ, or GSAKYQ. The PSA cleavage site is located atthe carboxy terminal side of Q.

Further examples of the peptides of the invention are constructed asanalogs of, derivatives of, and conservative variations on the aminoacids sequence SSKYQ, GKSQYQ, or GSAKYQ. Thus, the broader group ofpeptides having hydrophilic and hydrophobic substitutions, andconservative variations are encompassed by the invention. The term“isolated” as used herein refers to a peptide substantially free ofproteins, lipids, nucleic acids, for example, with which it is naturallyassociated. Those of skill in the art can make similar substitutions toachieve peptides with greater activity and/or specificity toward PSA.For example, the invention includes the peptide sequences describedabove, as well as analogs or derivatives thereof, as long as thebioactivity of the peptide remains. Minor modifications of the primaryamino acid sequence of the peptides of the invention may result inpeptides which have substantially equivalent activity as compared to thespecific peptides described herein. Such modifications may bedeliberate, as by site-directed mutagenesis or chemical synthesis, ormay be spontaneous. All of the peptides produced by these modificationsare included herein, as long as the biological activity of the originalpeptide remains, i.e., susceptibility to cleavage by PSA.

Peptides of the invention include any analog, homolog, mutant, isomer orderivative of the peptides disclosed in the present invention, as longas the bioactivity as described herein remains. All peptides weresynthesized using L-amino acids; however, D-forms of the amino acids canbe synthetically produced. In one embodiment, one or two of the serineresidues in the peptides of the invention are D-Serine residues.

The peptides of the invention include peptides which are conservativevariations of those peptides specifically exemplified herein. The term“conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tryptophan, tyrosine, norleucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. Neutral hydrophilic aminoacids which can be substituted for one another include asparagine,glutamine, serine, and threonine. The term “conservative variation” alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

A wide variety of groups can be linked to the carboxy terminus peptidesconsisting of or comprising SSKYQ, GKSQYQ, or GSAKYQ. Notably,therapeutic drugs can be linked to this position. In this way, advantageis taken of the PSA-specificity of the cleavage site, as well as otherfunctional characteristics of the peptides of the invention. Preferably,the therapeutic drugs are linked to the carboxy terminus either directlyor through a linker group. The direct linkage is preferably through anamide bond, in order to utilize the proteolytic activity and specificityof PSA. If the connection between the therapeutic drug and the aminoacid sequence is made through a linker, this connection is alsopreferably made through an amide bond, for the same reason. The linkermay be connected to the therapeutic drug through any of the bond typesand chemical groups known to those skilled in the art. The linker mayremain on the therapeutic drug indefinitely after cleavage, or may beremoved soon thereafter, either by further reactions with externalagents, or in a self-cleaving step. Self-cleaving linkers are thoselinkers which can intra molecularly cyclized and release the drug, orundergo spontaneous S_(N 1) solvolysis and release the drug upon peptidecleavage. Such linkers are for example 2, 2-dialkyl-2-(2-anisyl) aceticacid, described in Atwell et al., J. Med. Chem., 37:371-380, (1994), andp-amidobenzyloxycarbonyl, described in Carl et al., J. Med. Chem.,24:479-480, (1981). Further useful examples are provided in thesereferences. Other materials such as detectable labels or imagingcompounds can be linked to the peptide. Groups can also be linked to theamino terminus of the peptides described herein, including such moietiesas antibodies, and peptide toxins, including the 26 amino acid toxin,melittin and the 35 amino acid toxin, cecropin B, for example. Both ofthese peptide toxins have shown toxicity against cancer cell lines.

The length of the amino acid sequence plays a role in the ability of PSAto cleave the peptide, with at least a tetrapeptide required foractivity. Tetrapeptides as recited above typically are not as soluble ashexapeptides, although PSA cleavage activity is similar. One skilled inthe art will be able to readily identify specific groups to improve thewater solubility of the peptides of the invention. Among the groupswhich should be considered are polysaccharides, including dextrans,cyclodextrins, starches and the like, including derivatives thereof.Therapeutic drugs which are water soluble may be linked to the peptidesof the invention, thereby imparting water solubility to the complexes asa whole. The peptides of the invention may also contain conventionalcapping groups connected to the amino terminus of the peptide to preventendopeptidase activity from degrading the peptide. Such capping groupsinclude acetyl; succinyl, benzyloxycarbonyl, glutaryl,morpholinocarbonyl, and many others known in the art.

Amino-acid sequences can be constructed that contain highly specificcleavage sites for PSA. The highly PSA-specific cleavage sites of theinvention are cleaved by PSA to yield at least 5 picomoles of cleavedpeptide per minute per 200 picomoles of PSA. Preferably, the peptidescontain PSA-specific cleavage sites that yield at least 10 picomoles ofcleaved peptide per minute per 200 picomoles of PSA. Most preferably,such cleavage sites yield at least 15 picomoles of cleaved peptide perminute per 200 picomoles of PSA.

Amino acid sequences can be constructed that are highly selectivetowards cleavage by PSA, so that cleavage by other purifiedextracellular proteases is minimized. Preferably, the peptides of theinvention are cleaved by extracellular proteases other than PSA to yieldnot more than 4.0 picomoles of cleaved peptide per minute per 200picomoles of purified extracellular non-PSA proteases. More preferably,the peptides are cleaved to yield not more than 2.0 picomoles of cleavedpeptide per minute per 200 picomoles of purified extracellular non-PSAenzyme. Most preferably, not more than 2.0 picomole per minute ofpeptide are cleaved per 200 picomoles of purified extracellular non-PSAenzyme.

Highly PSA-specific amino acid sequences can be constructed that arealso stable toward cleavage in sera. Preferably, the peptides containingthis sequence yield at most 2.0 picomoles per minute of cleaved peptidein human serum. More preferably, the peptides containing this sequenceyield at most 1.75 picomoles per minute of cleaved peptide in humanserum. Most preferably, at most 1.5 picomoles per minute of cleavedpeptide are yielded by enzymes found in human serum.

The preferred amino acid sequences of the invention is also highlyselective towards cleavage by PSA as compared to purified intracellularproteases. Preferably, the peptides of the invention are cleaved byintracellular proteases other than PSA to yield not more than 35picomoles of cleaved peptide per minute per 200 picomoles of purifiedintracellular protease. More preferably, the peptide do not yield morethan 20 picomoles of cleaved peptide. Most preferably, not more than 5picomoles of cleaved peptide are produced upon cleavage by purifiedintracellular proteases other than PSA. While not wishing to be bound byany particular theory, it is believed that essentially no pathogeniceffects arise from cleavage of the peptides of the compositions of theinvention through intracellular proteases, and that these proteases donot play a significant role in the activation of the therapeutic drugsof the invention.

The peptides of the invention can be synthesized according to any of therecognized procedures in the art, including such commonly used methodsas t-boc or fmoc protection of alpha-amino groups. Both methods involvestepwise syntheses whereby a single amino acid is added at each stepstarting from the C-terminus of the peptide. (see, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well-knownsolid phase peptide synthesis methods described in Merrifield, J. Am.Chem. Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptideSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mM amine/gram polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼ to 1 hour at 0° C. After evaporation of the reagents, thepeptides are extracted from the polymer with 1% acetic acid solutionwhich is then lyophilized to yield the crude material. This can normallybe purified by such techniques as gel filtration on Sephadex G-15 using5% acetic acid as solvent. Lyophilization of appropriate fractions ofthe column will yield the homogeneous peptide of peptide derivatives,which can then be characterized by such standard techniques as aminoacid analysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by solid phase Edman degradation.

The invention encompasses isolated nucleic acid molecules encoding thepeptides of the invention, vectors containing these nucleic acidmolecules, cells harboring recombinant DNA encoding the peptides of theinvention, and fusion proteins which include the peptides of theinvention. Especially preferred are nucleic acid molecules encoding thepolypeptides described herein.

Prodrug Compositions

The invention also features prodrug compositions which comprise atherapeutic drug linked to a peptide as described herein, e.g., apeptide containing a cleavage site that is specific for prostatespecific antigen or any enzyme which has the enzymatic activity ofprostate specific antigen (PSA). As noted above, the peptides of theinvention can be used to activate therapeutic drugs at PSA producingtissue. The peptides which are useful in the prodrugs of the inventionare those described above.

The therapeutic drugs that may be used in the prodrugs of the inventioninclude any drugs which can be directly or indirectly linked to thePSA-specifically cleavable peptides of the invention. Preferred drugsare those containing primary amines. The presence of a primary amineallows the formation of an amide bond between the drug and the peptide.This bond serves as the cleavage site for PSA. The primary amines may befound in the drugs as commonly provided, or they may be added to thedrugs by chemical synthesis. The presence of the primary amine mustallow the therapeutic drug to retain its non-specific toxicity whencleaved. Certain therapeutic drugs contain primary amines, for example,anthracycline antibiotics containing an amino sugar such as doxorubicin,daunorubicin, epirubicin (4-epidoxorubicin), idarubicin(4-demethoxydaunomycin) and the like. These drugs intercalate intopolynucleotides and interfere with replication processes. Othertherapeutic drugs are required to have primary amines introduced bychemical or biochemical synthesis, for example, sesquiterpene-γ-lactonessuch as those belonging to the guaianolide, inuchineolide,germacranolide, and eudesmanolide families of sesquiterpenoids. Theseinclude estafiatin, grossheimin, inuchinenolide, arglabin, thapsigarginand their derivatives, such as thapsigargicin and many others known tothose skilled in the art. Thapsigargin and its derivatives are believedto act by inhibiting the SERCA pump found in many cells.

In an alternative embodiment, a peptide of the invention is linked to apolypeptide therapeutic. In a specific example, a peptide of theinvention can be linked to, for example, a bacterial toxin, e.g.,aerolysin, hemolysin, colicin, or diphtheria toxin. The bacterial toxinwould be inactive until the cleavage of the polypeptide by PSA, therebycreating a targeted cancer therapeutic.

The peptide and therapeutic drug are linked directly or indirectly (by alinker) through the carboxy terminus of the terminal amino acid residue.The site of attachment on the therapeutic drug must be such that thenon-specific toxicity of the drug is substantially inhibited. Thus, theprodrug should not be significantly toxic. In other words, the LC₅₀concentration of the therapeutic drug should be at least 5 times lowerthan the LC₅₀ concentration of the prodrugs of the invention, morepreferably at least 20 times lower, and most preferably the LC₅₀concentration of the therapeutic drug should be at least 100 times lowerthan the LC₅₀ concentration of the prodrugs of the invention.

In certain embodiments, the peptide and drug can be connected indirectlythrough a linker. The linker can either remain attached to the drug orbe cleaved off. In embodiments in which the linker remains attached tothe drug, the linker can be any group which does not substantiallyinhibit the non-specific toxicity of the drug after cleavage from thepeptide. Suitable linkers are primary amine containing alkanoyl,alkenoyl, and arenoyl substituents. Examples of such linkers areCO—(CH═CH)_(n1)—(CH₂)_(n2)—Ar—NH₂, CO—(CH₂)_(n2)—(CH═CH)_(n1)—Ar—NH₂,CO—(CH₂)_(n2)—(CH═CH)_(n1) —CO—NH—Ar—NH₂ andCO—(CH═CH)_(n1)—(CH₂)_(n2)—CO—NH—Ar—NH ₂ and substituted variationsthereof, where n1 and n2 are from 0 to 5, and Ar is any substituted orunsubstituted aryl group. Substituents which may be present on Arinclude short and medium chain alkyl, alkanoxy, aryl, aryloxy, andalkenoxy groups, nitro, halo, and primary secondary or tertiary aminogroups, as well as such groups connected to Ar by ester or amidelinkages. Amino acids can also serve as linkers.

In other embodiments, the linker is self-cleaving. Self-cleaving linkersare those which are disposed to cleave from the drug after the cleavageof the peptide by PSA. The linkers generally contain primary amineswhich form amide bonds to the carboxy terminus of the peptide sequence.The linkers can also contain a carboxylic acid which forms an amide bondto a primary amine found on the drug.

One method of linker self-cleavage relies on spontaneous S_(N1)solvolysis of the linker, activated by the cleavage of the peptide byPSA. The cleavage of the amide bond between the peptide terminalcarboxyl group and the primary amine on the linker releases π electrondensity into an aromatic system present in the linker, stabilizing thedevelopment of a positive charge developing on a carbon atom a to thearomatic system. This charge stabilization eliminates the carboxylicacid, which is subsequently hydrolyzed from the drug. Examples ofself-cleaving linkers of this type include p-amidobenzyloxycarbonyl, andsubstituted derivatives which do not significantly detrimentally affectthe stabilization of positive charge at the α carbon.

Another method of linker self-cleavage utilizes cyclization of aromaticamines substituted with alkanone groups which allow the formation ofintramolecular cyclic structures utilizing the lone electron pair of theamine which attack an electrophilic carbon such as that of the carbonyl.Five and six membered rings are formed preferably from such cyclization.Useful examples include 2-(2-anisyl) acetic acids and 3-(2-anisyl)propionic acids; as well as acid derivatives. With respect to suchderivatives, short chain alkyl groups such as methyl, ethyl are usefulas substituents. Naphthalene derivatives such as8-amino-1-naphthalenecarboxylic acid and 8-amino-1-naphthaleneaceticacid derivatives.

As outlined above, peptide cleavage frees the electrons of the amine,which attack the carbonyl carbon, allowing the drug (leaving group) tobe released. The carboxy terminus of the peptide is attached to theprimary amine group of the linker by an amide bond, and the primaryamine of the drug is attached to the carboxylic acid group of thelinker, also by an amide bond.

In such embodiments, the linker is not required to be non-interferingwith the non-specific toxicity of the drug, as long as it is cleavedwithin a period of time short enough to allow the drug to remainlocalized where it has been activated, or within a period of time shortenough to prevent inactivation by any means.

Preferably the prodrugs of the invention are not taken up by the cells,but are cleaved extracelullarly by PSA to yield at least 5 picomoles oftherapeutic drug per minute per 200 picomoles of PSA. Preferably, theprodrugs yield at least 10 picomoles of cleaved drug per minute per 200picomoles of PSA. Most preferably, at least 15 picomoles of cleaved drugper minute per 200 picomoles of PSA are produced.

Preferably, the prodrugs of the invention are cleaved by extracellularproteases other than PSA to yield not more than 4.0 picomoles of cleavedtherapeutic drug per minute per 200 picomoles of purified extracellularnon-PSA proteases. More preferably, the prodrugs are cleaved to yieldnot more than 2.0 picomoles of cleaved drug per minute per 200 picomolesof purified extracellular non-PSA enzyme. Most preferably, not more than2.0 picomole per minute of prodrug are cleaved per 200 picomoles ofpurified extracellular non-PSA enzyme.

Preferably, the prodrugs of the invention yield at most 2.0 picomolesper minute of cleaved therapeutic drug in human serum. More preferably,the prodrugs yield at most 1.75 picomoles per minute of cleaved drug inhuman serum. Most preferably, at most 1.5 picomoles per minute ofcleaved drug are yielded by enzymes found in human serum.

Preferably, the prodrugs of the invention are cleaved by intracellularproteases other than PSA to yield not more than 35 picomoles of cleaveddrug per minute per 200 picomoles of purified intracellular protease.More preferably, the prodrugs do not yield more than 20 picomoles ofcleaved drug. Most preferably, not more than 5 picomoles of cleaved drugare produced upon cleavage by purified intracellular proteases otherthan PSA. While not wishing to be bound by any particular theory, it isbelieved that essentially no pathogenic effects arise from cleavage ofthe peptides of the compositions of the invention through intracellularproteases, and that these proteases do not play a significant role inthe activation of the therapeutic drugs of the invention.

The prodrugs of the invention may also comprise groups which providesolubility to the prodrug as a whole in the solvent in which the prodrugis to be used. Most often the solvent is water. This feature of theinvention is important in the event that neither the peptide nor thetherapeutic drug is soluble enough to provide overall solubility to theprodrug. These groups include polysaccharides or other polyhydroxylatedmoieties. For example, dextran, cyclodextrin, starch and derivatives ofsuch groups may be included in the prodrug of the invention.

Sesquiterpene-γ-Lactone Analogs

The invention also features a derivatized sesquiterpene-γ-lactoneanalog, the derivatization including providing the molecule with aresidue substituted with a primary amine. The primary amine can be usedto link the derivatized sesquiterpene with various other moieties. Amongthese are peptides which link to the analog to give prodrugs withoutsignificant non-specific toxicity, but enzymatic reactions with PSAaffords the toxic drug. These enzymatic reactions can liberate thenon-specific toxic thapsigargin derivative, for example by cleavagethrough hydrolysis or proteolysis, various reactions of the side chainsof the peptide, or other reactions which restore the non-specifictoxicity of the sesquiterpene-γ-lactone derivative. These reactions canserve to activate the derivatized sesquiterpene locally at PSA-producingtissue, and with relative exclusivity to regions in which theseenzymatic is reactions take place. For example, if a derivatizedsesquiterpene-γ-lactone analog is linked, via a primary amine, to apeptide containing an amino acid sequence which includes a PSA-specificcleavage site, the analog can be released from the peptide selectivelyin regions where PSA, or other enzymes having the proteolytic activityof PSA, is found. The primary amine can likewise be used to link aderivatized sesquiterpene-γ-lactone analog to an antibody which binds anepitope in the target tissue.

Among the sesquiterpene-γ-lactone analogs preferred for use in thepresent invention are those of the guaianolide, inuchineolide,germacranolide, and eudesmanolide families of sesquiterpene-γ-lactoneanalogs. These include estafiatin, grossheimin, inuchinenolide,arglabin, thapsigargin and their derivatives, such as thapsigargicin andmany others known to those skilled in the art. One of the preferredclass of analogs is that based on the thapsigargin structure.

Thapsigargin is a sesquiterpene-γ-lactone having the following molecularstructure:

wherein R₁ and R₂ are substituents to be described below, and R₃ is analkanoyl or alkenoyl substituent, preferably angeloyl (CO—C(CH₃)═CHCH₃).

Thapsigargin is an effective inhibitor of the Ca²+ ion pump proteins ofintracellular membranes located in sacroplasmic reticulum (SR) andendoplasmic reticulum (ER) of skeletal, cardiac, muscle and brainmicrosomes. As such, it displays a general non-specific toxicity towardmany normal host cells. A method of targeting the proliferationindependent cytotoxicity of thapsigargin selectively to cancer cells isneeded. A number of thapsigargin analogs have been developed which canbe coupled to enzymatically susceptible moieties. The analogs of thepresent invention include thapsigargin analogs that contain primaryamines. The primary amines allow the coupling of thapsigargin analogs toappropriate moieties.

Referring to the thapsigargin skeleton, primary amines can be placed insubstituent groups pendant from either the C-2 or the C-8 carbon. Thesepositions are substituted with the groups -OR₁ and -OR₂ respectively inthe thapsigargin structure shown above. These substituent groups cancomprise primary amine-containing alkanoyl, alkenoyl or arenoylsubstituents. Preferably, these substituent groups are represented bythe following structures: unsubstituted or alkyl-, aryl-, halo-,alkoxy-, alkenyl-, amido- or amino-substituted CO—(CH═CH)_(n1)—(CH2)_(n2) —Ar—NH₂, CO—(CH₂)_(n2) —(CH═CH)_(n1) —Ar—NH₂ CO—(CH₂)_(n2)—(CH═CH)_(n1) —CO—NH—Ar—NH₂ and CO—(CH═CH)_(n1) —(CH₂)_(n2)—CO—NH—Ar—NH₂ and substituted variations thereof, where n1 and n2 arefrom 0 to 5, Ar is any substituted or unsubstituted aryl group, and theposition of NH₂ on Ar can be ortho, meta or para with respect to theposition of the remainder of the substituent group.

Preferred substituent groups are6-(N-[3-amino-4-methylphenyl]-carboxamido)hexanoyl,4-(N-[3-amino-4-methylphenyl]-carboxamido)butanoyl,3-(N-[3-amino-4-methylphenyl]-carboxamido)propanoyl, 4-aminobenzoyl,4-aminocinnamoyl, 3-[4-aminophenyl]propionoyl, 2-[4-aminophenyl]acetyl,and 4-[4-aminophenyl]butanoyl, 4-[4-aminophenyl]pentanoyl,4-[4-aminophenyl]hexanoyl, 4-[4-aminophenyl]heptanoyl, or4-[4-aminophenyl]ocanoyl substituents.

The primary amine-containing thapsigargin analogs as generally outlinedabove are synthesized by removing the 8-O-butanoyl group bytriethylamine catalyzed methanolysis of thapsigargin. Attachment ofanhydrides of dicarboxylic acids of various lengths affords analogs inwhich the acyl group attached to a 0-8 end in a free carboxylic acid. Adicyclohexylcarbodiimide (DCCI) promoted coupling of 2,4-diaminoarene tothe carboxylic acid analogs yield primary aromatic amine-containingthapsigargin analogs. The primary amine is a potential coupling pointfor a number of moieties; including the peptide sequence of theinvention, via the carboxyl terminus of the peptide.

The primary amine-containing thapsigargin analogs of the invention havenon-specific toxicity toward cells. This toxicity is measured as theconcentration of analog needed to kill 50% of clonogenic cells (LC₅₀).The LC₅₀ of the analogs of the invention is desirably at most 20 μM,preferably at most 5 μM, and more preferably at most 500 nM of analog.

The primary amine-containing thapsigargin analogs of the invention haveendoplasmic reticulum Ca²⁺ -ATPase inhibitory activity. This activity ismeasured as the concentration of analog needed to inhibit 50% of thisATPase (IC₅₀). The IC₅₀ of the analogs of the invention is desirably atmost 500 nM, preferably at most 200 nM, and more preferably at most 50nM of analog.

A preferred thapsigargin molecule is8—O-(12-[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT).

Methods of Treatment Using Prodrugs

The invention also provides methods of treating PSA-producing cellproliferative disorders of the invention with the prodrugs of theinvention.

The prodrugs of the invention and/or analogs or derivatives thereof canbe administered to any host, including a human or non-human animal, inan amount effective to treat a disorder.

The prodrugs of the invention can be administered parenterally byinjection or by gradual infusion over time. The prodrugs can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. Preferred methods fordelivery of the prodrug include intravenous or subcutaneousadministration. Other methods of administration will be known to thoseskilled in the art.

Preparations for parenteral administration of a prodrug of the inventioninclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer, sdextrose), and the like. Preservatives and other additives can also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases, and the like.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. ThePSA-specific prodrugs of the invention are useful in treatingmalignancies of the various organ systems. Essentially, any disorderwhich is etiologically linked to PSA expression could be consideredsusceptible to treatment with a PSA-specific prodrug. One such disorderis a malignant cell proliferative disorder, for example. The term“therapeutically effective amount” as used herein for treatment of cellproliferative disorders refers to the amount of prodrug sufficient tocause a reduction in the number of unwanted cells. The term“therapeutically effective” therefore includes the amount of prodrugsufficient to prevent, and preferably reduce by at least 25%, and morepreferably to reduce by 90%, the number of unwanted cells. The dosageranges for the administration of prodrug are those large enough toproduce the desired effect. Generally the dosage will vary with age,condition, sex, and extent of the disorder in the subject, and can bedetermined by one skilled in the art. The dosage can be adjusted by theindividual physician in the event of any contraindications. In anyevent, the effectiveness of treatment can be determined by monitoringtumor ablation. For non-malignant conditions such as benign prostatichyperplasia the effectiveness of therapy can be monitored clinically bydecrease in the size of the prostate on digital rectal exam. Inaddition, effectiveness can also be measured by evaluating urinaryparameters such as voiding frequency, urgency and number of episodes ofurination at night (i.e. nocturia).

Method of Producing Prodrugs

The invention, in another aspect, provides a method of producing theprodrugs of the invention. This method involves linking atherapeutically active drug to a peptide of the invention. After thedrug and peptide are linked to produce a therapeutic prodrugcomposition, the non-specific toxicity of the drug is substantiallyinhibited. In certain embodiments, the peptide is linked directly to thedrug. In other embodiments, the peptide is indirectly linked to thedrug, the linkage occurring through a linker. In each case the carboxyterminus of the peptide is used for linking. The therapeutic drugcontains a primary amine group to facilitate the formation of an amidebond with the peptide. Many acceptable methods of coupling carboxyl andamino groups to form amide bonds are known to those of skill in the art.

This bond is cleaved by PSA, releasing the therapeutic drug. Suitablelinkers include any chemical group which contain a primary amine. Thelinkers for use in the present invention include amino acids, primaryamine-containing alkyl, alkenyl or arenyl groups.

The connection between the linker and the therapeutic drug may be of anytype known in the art, preferably covalent bonding. The linker group mayremain attached to the therapeutic drug if its attachment does notsignificantly reduce the non-specific toxicity of the drug. In certainembodiments, the linker is a cleavable linker, which may be cleavedeither by an external agent, or it may be a self-cleaving linker.External agents which may effect cleavage of the linker include enzymes,proteins, organic or inorganic reagents, protons and any other agentswhich do not affect the non-specific toxicity of the drug or prodrug.

In certain embodiments, the linker comprises an amino acid sequence. Thesequence may be of any length, but is preferably between 1 and 10 aminoacids, most preferably between 1 and 5 amino acids in length. Preferredamino acids are leucine, histidine, or amino acid sequences containingthese amino acids, especially at their amino termini, althoughconservative variations of these amino acids may also be utilized.

Other groups may be added to the prodrugs of the invention, includingthose which render the prodrug soluble in water. These groups includepolysaccharides or other polyhydroxylated moieties. For example,dextran, cyclodextrin and starch may be included in the prodrug of theinvention.

Method-of Screening Tissue

In another aspect the invention provides a method of detectingPSA-producing tissue using the peptides of the invention, as describedabove. The method is carried out by contacting a detectably labeledpeptide of the invention with target tissue for a period of timesufficient to allow PSA to cleave the peptide and release the detectablelabel. The detectable label is then detected. The level of detection isthen compared to that of a control sample not contacted with the targettissue. Many varieties of detectable label are available, includingoptically based labels, such as chromophoric, chemiluminescent,fluorescent or phosphorescent labels, and radioactive labels, such asalpha, beta or gamma emitting labels. Examples of fluorescent labelsinclude amine-containing coumarins such as 7-amino-4-methylcoumarin,7-amino-4-trifluoromethyl, and other amine-containing fluorophores suchas 6-aminoquinoline, and rhodamines, including rhodamine. Examples ofradioactive labels include beta emitters such as ³ H, ¹⁴ C and ¹²⁵ I.Examples of chromophoric labels (those that have characteristicabsorption spectra) include nitroaromatic compounds such asp-nitroaniline. Examples of chemiluminescent labels include luciferinssuch as 6-amino-6-deoxyluciferin.

Preferably, the choice of detectable label allows for rapid detectionand easily interpretable determinations. Detectable labels for use inthe invention preferably show clearly detectable differences betweendetection from the cleaved and uncleaved state.

The invention provides a method for detecting a cell proliferativedisorder which comprises contacting a PSA-specific peptide with a cellsuspected of having a PSA-production associated disorder and detectingcleavage of the peptide. The peptide reactive with PSA is labeled with acompound which allows detection of cleavage by PSA. For purposes of theinvention, a peptide specific for PSA may be used to detect the level ofenzymatically active PSA in biological fluids and tissues such assaliva, blood, or urine. Any specimen containing a detectable amount ofantigen can be used. The level of PSA in the suspect cell can becompared with the level in a normal cell to determine whether thesubject has a PSA-production associated cell proliferative disorder.Preferably the subject is human.

Method of Screening Prodrugs

The invention also provides a method of selecting potential prodrugs foruse in the invention. The method generally consists of contactingprodrugs of the invention with PSA-producing tissue and non-PSAproducing tissue in a parallel experiment. “PSA-producing tissue” asused herein is tissue that produces at least 1 ng enzymatically activePSA/mL of fluid from tissue, or at least 1 ng of enzymatically activePSA/10⁶ cells/24 hours from cells. The prodrugs which exert toxiceffects in the presence of PSA-producing tissue, but not in the presenceof non-PSA producing tissue are suitable for the uses of the invention.In other words, the LC₅₀ concentration of the prodrug in the presence ofPSA-producing tissue is at least 3 times lower than the LC₅₀concentration of the prodrug in the presence of non-PSA producingtissue, more preferably at least 20 times lower, and most preferably theLC₅₀ concentration of the prodrug in the presence of PSA-producingtissue is at least 100 times lower than the LC₅₀ concentration of theprodrug in the presence of non-PSA producing tissue.

Method of Determining PSA Activity

The invention also provides a method of determining the activity of PSA.The method generally consists of contacting detectably labeled prodrugsof the invention with samples may come from fluid drawn fromPSA-producing tissue, from tissue culture media, from serum, saliva orurine, or any source which contains PSA. The cleavage of peptide whichtakes place by PSA results in the release of a detectable label, whichis subsequently detected. This detection level is compared to thedetection level which is found upon performing a parallel experiment inwhich the PSA-containing sample is a standard solution made up frompurified PSA as described, for example, in Christensson, et al., Eur. J.Biochem. 194:755-765, (1990). This comparison results in a determinationof the activity of the PSA which is present in the sample, given acorrection for any differences in PSA concentration which may exist.Such correction may be accomplished directly by adjusting theconcentrations of the standard and sample solutions to match each otheror by mathematical correction means.

Method of Imaging Tissue

The invention in another aspect, provides a method of imaging softtissue or bone metastases by providing peptides of the invention linkedto lipophilic imaging labels that can be detected by imaging techniques,for example, positron emission tomography (PET). This method isaccomplished generally by administering a peptide of the inventionlinked to a primary amine-containing lipophilic label to a subjecthaving or suspected of having a PSA-producing associated cellproliferative disorder. The peptide is selectively cleaved from thelipophilic imaging label where enzymatically active PSA occurs in thesubject (i.e., PSA producing tissues). The lipophilic imaging label isthen drawn into the membranes of cells in the vicinity. After a periodof time sufficient to allow cleavage of the peptide by PSA, and to allowthe uncleaved peptide to be sufficiently cleared from the subject toallow reliable imaging, the subject is imaged. The lipophilic labelaccumulates in the soft tissue or bone that produces PSA, and allows adiagnosis of the subject. Suitable labels for PET scanning areradionuclides such as ¹⁸ F, ¹¹ C, ¹³ N and ¹⁵ O, and any other positronemitters known in the art. Lipophilicity can be engineered into thelabel by introducing the label into lipophilic fragments or moietiesknown to those in the art, by methods known to those skilled in the art.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

The following examples illustrate the preparation and properties ofcertain embodiments of the invention. The goal of the followingexperiments was to identify a PSA-cleavable peptide that had improvedpharmacological properties when used in a PSA-activated pro-drug.

Example 1

The development of a PSA-activated pro-drug requires advancements on twofronts: 1) the identification of an optimal peptide sequence to serve asa highly active PSA substrate while being stable to cleavage by otherproteases in the body and 2) identification of a thapsigargin derivativethat could be chemically coupled to a PSA-substrate peptide, yet retainall the activity of the parent thapsigargin after it was released fromthe prodrug by PSA activity. These combined efforts resulted inidentification of an initial PSA-activated pro-drug which was identifiedusing strategies and systematic screenings that are described in detailelsewhere (see, for example, U.S. Pat. No. 6,410,514). This prodrugcontained the peptide sequence Mu-His-Ser-Ser-Lys-Leu-Gln (Mu-HSSKLQ).This peptide was initially coupled to a fluorophore, 7-amino-4-methylcoumarin to generate the fluorescent PSA substrate Mu-HSSKLQ-AMC. Thissubstrate was then used to determine enzyme kinetics of PSA hydrolysis.Subsequently this peptide was coupled to the L12ADT active drug togenerate the prodrug Mu-HSSKLQ//L12ADT (where // indicates site of PSAcleavage).

Subsequently, a series of studies was initiated to identify a moreoptimal PSA cleavable peptide. The first study involved synthesis of aseries of putative PSA substrates based on the starting sequence GSSKLQ.In brief, this technology involved synthesis of small amount offluorescent peptide onto cellulose membrane. Addition of PSA to thecellulose membrane containing well resulted in release of fluorescenttag over time. Release rate was dependent on affinity of peptide forPSA. At defined time points, aliquots were removed and tested for totalfluorescence in separate plate. The assay allowed for measurement ofhydrolysis over 4 time points. From this data a hydrolysis rate wasdetermined for each peptide as summarized below in Tables 1 and 2

TABLE 1 Hydrolysis of selected putative PSA peptides by PSA (10 μg/ml)

Most preferred peptide sequence indicated in bold lettering. D-aminoacids in lower case type.

In this first set of peptides, hydrolysis of the test peptide GSSKLQ wascompared to a series of peptides modified in various positions. One typeof analysis involved an “alanine scan” in which each position of thepeptide was replaced by alanine in an attempt to reveal which aminoacids are most critical for PSA activity. This scan revealed that thelysine (K) in P3, the leucine (L) in P2 and glutamine (Q) in P1 positionwere important factors mediating PSA hydrolysis. In contrast, theserines in positions P4 and P5 could be replaced resulting in enhancedPSA hydrolysis.

A second type of analysis involved substitution of D-amino acids fornative L-amino acids in the peptide. This type of analysis is performedto identify amino acids that could be changed to D-configuration in anattempt to make a peptide that is more stable to non-specific hydrolysisby serum proteases. This analysis revealed that D-amino acidsubstitution in positions P-1, and P1-P3 resulted in significantreduction in PSA hydrolysis whereas D-amino acid substitution could betolerated in positions P4 and P5 of the peptide.

In the next set of studies a series of peptides were synthesized, someof which were based on substitutions in the test GSSKLQ peptide and someof which were based on rearrangement of the sequences in the testpeptide, Table 2.

TABLE 2 Hydrolysis of additional peptides by PSA (10 μg/ml)

This analysis revealed unexpectedly that the most significant changethat augmented PSA activity was the substitution of the amino acidtyrosine (Y) for leucine (L) in the P2 position of the peptide. Theresults also confirmed the requirement for lysine (K) in either the P3or P5 position of the peptide.

On the basis of these results a series of putative fluorescent PSAsubstrates were synthesized by coupling 7-amino-4-mehtyl coumarin to theC-terminal carboxyl group of the peptides. These peptides were thenincubated with PSA (10 μg/ml) and Michaelis Menton kinetic parametersdetermined using Lineweaver Burke plots, Table 3. In this study,K_(cat)/K_(m) ratios were calculated in order to rank peptides. Newlysynthesized peptides contained an acetyl protecting group (Ac) at theamino terminus, whereas the test peptide contained the protecting groupmorpholinylcarbonyl (Mu).

Table 3. Kinetic parameter of flourescent PSA peptides

Km k_(cat) k_(cat)/Km Peptide Sequence (μM) (l/s) (s⁻¹ M⁻¹) 1.Ac-GKSQYQ- 343 0.09 278 AMC 2. Ac-GSSKYQ- 500 0.113 227 AMC 3.Ac-GsAKYQ- 653 0.143 218 AMC 4. Ac-GSSKFQ- 666 0.057 86 AMC 5.Mu-HSSKLQ-AMC 470 0.011 23.6

These analyses revealed that peptides containing tyrosine (Y) in the P2position had approximately equal Kcat/Km ratios that were 10-fold higherthan the Mu-HSSKLQ-AMC test peptide. In each case this increasedactivity appeared to be due to ˜10-fold improvement in the k_(cat)kinetic parameter.

On this basis, the peptide with the sequence GSSKYQ was selected forfurther study. For further study, the glycine at the amino terminal endwas eliminated from the peptide. In addition, the initial Ac protectinggroup was replaced by the more water soluble morpholinocarbonyl (Mu)group. Using this rationale, the prodrug Mu-SSKYQ//L12ADT wassynthesized, FIG. 1.

Subsequently PSA hydrolysis of the new prodrug Mu-SSKYQ//L12ADT wascompared head to head with the test prodrug Mu-HSSKLQ//L12ADT.Unexpectedly, the new prodrug was hydrolyzed dramatically better thanthe test peptide, FIG. 2.

The antitumor efficacy of Mu-SSKYQ//L12ADT was compared toMu-HSSKLQ//L12ADT at equimolar intravenous dose of ˜7 mg/kg/day (i.e.0.1 μmole/dose/day) for 10 consecutive doses, FIG. 3. For these studieswe generated a high PSA producing variant of CWR22 through passage incastrate hosts (i.e. CWR22H). This model allows us to assess directantitumor effects of these prodrugs without concerns that the drugs maylower serum testosterone. In this study, the Mu-HSSKLQ//L12ADT prodrughad a Tumor Volume/Control Volume (i.e. T/C ratio) of 0.57 after 10 daysof therapy whereas the Mu-SSKYQ//L12ADT had a significantly better T/Cratio of 0.41. In addition, none of the animals in the Vehicle controlor the Mu-HSSKLQ//L12ADT treatment group showed tumor regression,whereas 33% of animals in the Mu-SSKYQ//L12ADT treated group hadregression of tumors from starting size after 10 days of therapy.

In a subsequent study, CWR22H bearing animals were treated for 10 dayswith either Mu-HSSKLQ//L12ADT or Mu-SSKYQ//L12ADT over 3 orders ofmagnitude of dose levels (i.e. 0.1, 0.01 and 0.001 μmole/dose/day).Twenty four hours after the last dose (i.e. trough), tumors wereharvested, homogenized and trough levels of prodrug and free L12ADTdetermined using LC/MS analysis, FIG. 4.

The disclosed results demonstrate that at each dose: level,comparatively higher amounts of the active drug L12ADT are released fromthe Mu-SSKYQ//L12ADT prodrug than from the Mu-HSSKLQ//L12ADT prodrug.These results are consistent with the in vitro biochemical studiesshowing enhanced cleavage of the Mu-SSKYQ//L12ADT prodrug by PSA.

In summary the results presented above document that theMu-SSKYQ//L12ADT prodrug represents a significant improvement over theprevious Mu-HSSKLQ//L12ADT prodrug in terms of PSA hydrolysis, antitumorefficacy against PSA producing human prostate cancer xenografts andgeneration of active L12ADT drug within the tumors.

Incorporation by Reference

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A composition comprising a prodrug, the prodrug comprising:8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT); and a peptide comprising sequence Ser-Ser-Lys-Tyr-Gln (SEQ IDNO:1); wherein the peptide is linked to the L12ADT to inhibit thetherapeutic activity of the drug, and wherein the L12ADT is cleaved fromthe peptide upon proteolysis by an enzyme having a proteolytic activityof prostate specific antigen (PSA).
 2. A prodrug comprising:8-O-(12-[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT); and a peptide comprising the sequence Ser-Ser-Lys-Tyr-Gln (SEQID NO: 1); wherein the peptide is linked to the L12ADT to inhibit thetherapeutic activity of the drug, and wherein the L12ADT is cleaved fromthe peptide upon proteolysis by an enzyme having a proteolytic activityof prostate specific antigen (PSA).
 3. A prodrug comprising:8-O-(12-[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT); and a peptide consisting of the sequence Ser-Ser-Lys-Tyr-Gln(SEQ ID NO:1); wherein the peptide is linked to the L12ADT to inhibitthe therapeutic activity of the drug, and wherein the L12ADT is cleavedfrom the peptide upon proteolysis by an enzyme having a proteolyticactivity of prostate specific antigen (PSA).
 4. The composition of claim1, or claim 2, wherein the peptide consists of the sequenceSer-Ser-Lys-Tyr-Gln (SEQ ID NO: 1).
 5. The composition of claim 1,wherein the peptide is linked directly to8-O-(12-[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT).
 6. The composition of claim 1, wherein the peptide is linkedto 8-O-(12-[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin(L12ADT) via a moiety comprising a primary amino group.
 7. Thecomposition of claim 6, wherein the peptide is linked to thapsigargin ora thapsigargin derivative via a linker associated with the primary aminogroup.
 8. The composition of claim 7, wherein the linker is an aminoacid sequence.
 9. The composition of claim 8, wherein the linkercomprises a leucine residue.
 10. The composition of claim 7, wherein thelinker is selected from the group consisting of unsubstituted or alkyl-,aryl-, halo-, alkoxy-, alkenyl-, amido-, or amino-substitutedCO—(CH═CH)_(n1)—(CH₂)_(n2)—Ar—NH₂, CO—(CH₂)_(n2)—(CH═CH)_(n1)—Ar—NH₂,CO—(CH₂)_(n2)—(CH═CH)_(n1)—CO—NH—Ar—NH₂ andCO—(CH═CH)_(n1)—(CH.sub.2)_(n2)—CO—NH—Ar—NH₂ wherein n1 and n2 are from0 to 5, Ar is any substituted or unsubstituted aryl group, andattachment of NH₂ to Ar is in a ortho, meta or para position withrespect to the remainder of the linker.
 11. The composition of claim 1,further comprising an added substituent which renders the compositionwater soluble.
 12. The composition of claim 11, wherein the addedsubstituent is a polysaccharide.
 13. The composition of claim 12,wherein the polysaceharide is selected from the group consisting ofmodified or unmodified dextran, cyclodextrin and starch.
 14. A method oftreating a PSA-producing cell proliferative disorder, the methodcomprising administering the composition of claim 1 in a therapeuticallyeffective amount to a subject having the cell proliferative disorder.15. The method of claim 14, wherein the disorder is benign.
 16. Themethod of claim 15, wherein the benign disorder is benign prostatehyperplasia.
 17. The method of claim 14, wherein the disorder ismalignant.
 18. The method of claim 17, wherein the malignant disorder isprostate cancer.
 19. The method of claim 18, wherein the malignantdisorder is breast cancer.